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. 2010 Mar 19;285(12):9067-76.
doi: 10.1074/jbc.M109.070003. Epub 2010 Jan 21.

Expression of a homeostatic regulator, Wip1 (wild-type p53-induced phosphatase), is temporally induced by c-Jun and p53 in response to UV irradiation

Ji-young Song  1 , Hye-Sook Han, Kanaga Sabapathy, Byung-Moo Lee, Eunsil Yu, Jene Choi
Affiliations

Expression of a homeostatic regulator, Wip1 (wild-type p53-induced phosphatase), is temporally induced by c-Jun and p53 in response to UV irradiation

Ji-young Song et al. J Biol Chem. .

Abstract

Wild-type p53-induced phosphatase (Wip1) is induced by p53 in response to stress, which results in the dephosphorylation of proteins (i.e. p38 MAPK, p53, and uracil DNA glycosylase) involved in DNA repair and cell cycle checkpoint pathways. p38 MAPK-p53 signaling is a unique way to induce Wip1 in response to stress. Here, we show that c-Jun directly binds to and activates the Wip1 promoter in response to UV irradiation. The binding of p53 to the promoter occurs earlier than that of c-Jun. In experiments, mutation of the p53 response element (p53RE) or c-Jun consensus sites reduced promoter activity in both non-stressed and stressed A549 cells. Overexpression of p53 significantly decreased Wip1 expression in HCT116 p53(+/+) cells but increased it in HCT116 p53(-/-) cells. Adenovirus-mediated p53 overexpression greatly decreased JNK activity. Up-regulation of Wip1 via the p38 MAPK-p53 and JNK-c-Jun pathways is specific, as demonstrated by our findings that p38 MAPK and JNK inhibitors affected the expression of the Wip1 protein, whereas an ERK inhibitor did not. c-Jun activation occurred much more quickly, and to a greater extent, in A549-E6 cells than in A549 cells, with delayed but fully induced Wip1 expression. These data indicate that Wip1 is activated via both the JNK-c-Jun and p38 MAPK-p53 signaling pathways and that temporal induction of Wip1 depends largely on the balance between c-Jun and p53, which compete for JNK binding. Moreover, our results suggest that JNK-c-Jun-mediated Wip1 induction could serve as a major signaling pathway in human tumors in response to frequent p53 mutation.

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Figures

FIGURE 1.
FIGURE 1.
c-Jun binds to and activates the Wip1 promoter in A549 cells. A, Wip1 mRNA transcripts were analyzed via qRT-PCR after UV irradiation (50 J/m2) for the indicated times. Data indicate the level of Wip1 transcripts relative to a control gene, GAPDH. The mean of three experiments is shown in each column, and bars correspond with the S.D. B, cells were transfected with the Wip1 reporter plasmid Wip1–543-Luc or the p53RE-containing reporter plasmid (p53-induced Luc, as a control), together with a Renilla luciferase vector. After 8, 16, or 24 h of UV irradiation, luciferase activity was measured and normalized to control Renilla luciferase units. C, Wip1–543-Luc constructs were co-transfected with the control plasmid, ATF-2, c-Jun, or a combination of c-Jun and c-Fos. After 48 h of UV irradiation, Wip1 promoter activity was determined as described in B. D, cells were co-transfected with Wip1–543-Luc and the control plasmid, c-Jun, or a transactivation domain-deficient c-Jun mutant (TAM-67) and subjected to UV irradiation. After 48 h, cell extracts were assayed for luciferase activity. The mean of three experiments is shown in each column, and bars correspond with the S.D. *, p < 0.05, according to a two-tailed Student's t test.
FIGURE 2.
FIGURE 2.
c-Jun binds to the Wip1 promoter after UV irradiation in vitro. A, EMSA was performed using a 32P-labeled wild-type oligonucleotide containing the c-Jun binding site on the Wip1 promoter. The EMSA was performed using A549 nuclear extracts (NEs) prepared after 6 h of UV irradiation (50 J/m2). Competition assays were performed using 1-fold, 10-fold, and 100-fold molar excesses of cold wild-type (wt) or mutant (mt) oligonucleotides. B, for supershift assays, phospho-c-Jun (Ser-73) or control IgG antibodies (1 μg) were preincubated with the nuclear extracts.
FIGURE 3.
FIGURE 3.
p53 overexpression inhibits the Wip1 promoter. A and B, HCT116 p53+/+ and HCT116 p53−/− cells were transfected with Wip1–543-Luc having plasmids encoding c-Jun, p53, or a combination of the two proteins for 24 h. Cells were then exposed to UV irradiation (50 J/m2). Luciferase activity was measured after 48 h. C, HCT116 p53+/+ cells were co-transfected with Wip1–543-Luc and the indicated amount of a p53-encoding plasmid for 24 h. The total amount of transfected DNA was normalized to that of an empty plasmid. After 48 h of UV irradiation, luciferase activity was measured. The mean of three experiments is shown in each column, and bars correspond with the S.D. *, p < 0.05. D, HCT116 p53+/+ cells were infected with a control adenovirus encoding LacZ or an adenovirus encoding p53 at an MOI of 50 for 8 h and then exposed to UV irradiation. Then 16 h later, the cells were subjected to in vitro kinase assays with GST-c-Jun and immunoblot analysis using antibodies specific to p53 and phospho-c-Jun (Ser-73).
FIGURE 4.
FIGURE 4.
c-Jun and p53 regulate Wip1 expression in a time-dependent manner. A, ChIP analysis of p53 and c-Jun binding to the Wip1 promoter was performed in A549 cells. After exposure to UV irradiation for the indicated times, cells were prepared for ChIP with p53, c-Jun, or control IgG antibodies. B and C, A549 cells were transiently transfected with vectors encoding c-Jun or p53 for 24 h, then exposed to UV irradiation (50 J/m2). Cells were processed for ChIP with p53 or c-Jun antibodies after the exposure to UV irradiation for the indicated times.
FIGURE 5.
FIGURE 5.
c-Jun can substitute for p53 function in Wip1 expression. A and B, embryo fibroblasts of c-Jun+/+ and c-Jun−/− were transfected with Wip1–543-Luc encoding plasmids of c-Jun, p53, or a combination of the two proteins for 24 h. Fibroblasts were then exposed to UV irradiation (50 J/m2). Luciferase activity was measured after 48 h. C, A549 cells were transfected for 24 h with Wip1–543-Luc, Wip1-c-Jun-mut (i.e. containing a mutated c-Jun binding site), Wip1-p53-mut (i.e. containing a mutated p53RE binding site), or double-mut (i.e. containing mutated c-Jun and p53 RE binding sites) and then exposed to UV irradiation. After 48 h, luciferase assays were performed. D, A549 cells were transfected with Wip1-p53-mut with increasing amounts of c-Jun-encoding plasmids (10, 100, 200, 500, and 1000 ng) or Wip1–543-Luc as a control and then assayed as described in C. The mean of three experiments is shown in each column, and bars correspond with the S.D. *, p < 0.05.
FIGURE 6.
FIGURE 6.
MAPKs specifically activate Wip1 expression. A, A549 cells were transfected with Wip1–543-Luc and then pretreated for 1 h with the JNK-specific inhibitor SP600125 (20 μm), the ERK-specific inhibitor U0126 (10 μm), or the p38-specific inhibitor SB202190 (20 μm). After 48 h of UV irradiation, cell extracts were assayed for luciferase activity. The mean of three experiments is shown in each column, and bars correspond with the S.D. *, p < 0.05. B, A549 cells were pretreated with MAPK inhibitors as described in A and then exposed to UV irradiation. After 48 h, total cell lysates were prepared and subjected to immunoblotting assays using p53, phospho-p53 (Ser-43), c-Jun, phospho-c-Jun (Ser-73), and Wip1 antibodies. β-Actin was used as the loading control. C, A549 cells were transfected with either c-Jun siRNA or control siRNA for 48 h and exposed to UV irradiation. The cells were cultured for 24 h and subjected to immunoblotting assays using antibodies specific to c-Jun, phospho-c-Jun (Ser-73), Wip1, and β-actin. β-Actin was used as a loading control.
FIGURE 7.
FIGURE 7.
Wip1 is induced in both A549 (p53+/+) and A549-E6 (p53-null phenotype) cells. A549 (left) and A549-E6 (right) cells were exposed to UV irradiation (50 J/m2) and harvested at the times indicated. Western blot analysis was performed using specific antibodies against phosphorylated JNK, c-Jun, p38, p21, and Wip1. Expression of β-actin is shown as a loading control.
FIGURE 8.
FIGURE 8.
Proposed model for the temporal regulation of the Wip1 promoter by the p38 MAPK-p53 and JNK-c-Jun pathways. After UV irradiation, p53 is immediately activated by various kinases and then induces the Wip1 promoter. Later, p53 is dephosphorylated or inactivated by negative regulators such as Mdm2 and Wip1, and the JNK-c-Jun pathway thus ultimately becomes the major activator of the Wip1 promoter.
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